HIV-1 splicing is controlled by local RNA structure and binding of splicing regulatory proteins at the major 5′ splice site

Müeller, Nancy; Berkhout, Ben; Das, Atze T.

doi:10.1099/vir.0.000122

Cited by 18 publications

(25 citation statements)

References 59 publications

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“…Our data suggest that the SLSA1 structure is important to A1 usage; other splice acceptors and splice control elements are also located in or near structural features, and many of these regions have been studied for their ability to bind cellular splice factors (49,50). It will be informative to continue to characterize the roles that these and other structures play in splicing regulation by creating mutations that alter either structure or cellular factor binding sites.…”

Section: Discussionmentioning

confidence: 87%

Characterizing HIV-1 Splicing by Using Next-Generation Sequencing

Emery

Zhou

Pollom

et al. 2017

J Virol

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Full-length human immunodeficiency virus type 1 (HIV-1) RNA serves as the genome or as an mRNA, or this RNA undergoes splicing using four donors and 10 acceptors to create over 50 physiologically relevant transcripts in two size classes (1.8 kb and 4 kb). We developed an assay using Primer ID-tagged deep sequencing to quantify HIV-1 splicing. Using the lab strain NL4-3, we found that A5 (/) is the most commonly used acceptor (about 50%) and A3 () the least used (about 3%). Two small exons are made when a splice to acceptor A1 or A2 is followed by activation of donor D2 or D3, and the high-level use of D2 and D3 dramatically reduces the amount of and transcripts. We observed distinct patterns of temperature sensitivity of splicing to acceptors A1 and A2. In addition, disruption of a conserved structure proximal to A1 caused a 10-fold reduction in all transcripts that utilized A1. Analysis of a panel of subtype B transmitted/founder viruses showed that splicing patterns are conserved, but with surprising variability of usage. A subtype C isolate was similar, while a simian immunodeficiency virus (SIV) isolate showed significant differences. We also observed transsplicing from a downstream donor on one transcript to an upstream acceptor on a different transcript, which we detected in 0.3% of 1.8-kb RNA reads. There were several examples of splicing suppression when the intron was retained in the 4-kb size class. These results demonstrate the utility of this assay and identify new examples of HIV-1 splicing regulation. During HIV-1 replication, over 50 conserved spliced RNA variants are generated. The splicing assay described here uses new developments in deep-sequencing technology combined with Primer ID-tagged cDNA primers to efficiently quantify HIV-1 splicing at a depth that allows even low-frequency splice variants to be monitored. We have used this assay to examine several features of HIV-1 splicing and to identify new examples of different mechanisms of regulation of these splicing patterns. This splicing assay can be used to explore in detail how HIV-1 splicing is regulated and, with moderate throughput, could be used to screen for structural elements, small molecules, and host factors that alter these relatively conserved splicing patterns.

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Section: Discussionmentioning

confidence: 87%

Characterizing HIV-1 Splicing by Using Next-Generation Sequencing

Emery

Zhou

Pollom

et al. 2017

J Virol

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“…The virus has developed strategies to maintain transcript homeostasis by using cis RNA sequence elements that recruit host RNA-binding proteins to modulate the activities of donor and acceptor sites. The series of events that control HIV-1 splicing are not well understood; however, there is growing precedence that RNA structure modulates HIV-1 splicing (46,47). Therefore, an understanding of mechanisms that determine how frequently a splice site gets utilized requires accurate knowledge of the RNA interactions surrounding it.…”

Section: Discussionmentioning

confidence: 99%

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Jain

Morgan

Rife

et al. 2016

Journal of Biological Chemistry

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Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3 acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.Human immunodeficiency virus type 1 (HIV-1) 2 requires controlled synthesis of its protein complement for persistent infection and successful virion production. Genome expression is tightly regulated at the levels of transcription, splicing, mRNA nuclear export, and translation (1). RNA polymerase II-dependent transcription yields a 9-kilobase (kb) polycistronic transcript that undergoes multiple rounds of alternative splicing to produce upward of 100 different viral mRNAs that are classified by their intron composition: unspliced, incompletely spliced (4 kb), and completely spliced (2 kb) (2, 3). During early phase HIV-1 replication, cytoplasmic levels of the 2-kb transcripts predominate to encode Tat, Rev, and Nef. Accumulation of unspliced and 4-kb transcripts coincides with the transition to late phase replication and expression of Gag, Gag/Pol, Vpr, Vif, and Env/Vpu. Thus, HIV-1 splicing pathways are essential components of the viral replication cycle and represent new targets for therapeutic intervention (3, 4).The identities of HIV-1 transcripts are determined by the combinatorial use of several 5Ј donor and 3Ј acceptor splice sites. Efforts to understand mechanisms of HIV-1 splicing reveal that all of the acceptor sites and splice donors D2-D4 are suboptimal due to non-consensus core splicing signals (4, 5). Proper spliced ratios are therefore established via auxiliary cis RNA features, collectively known as splicing regulatory elements. Splicing regulatory elements function either as enhancers or silencers of splicing by recruiting host proteins belonging to the serine-arginine-rich (SR) and hnRNP families, respectively. Splicing regulatory element location (intronic or exonic) influences how frequently a given splice site is utilized, thereby making it difficult to determine general rules of the mechanisms that regulate alternative splicing (6).Splicing from site D4 to A7 removes the Rev-responsive element, which leads to the accu...

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“…The analysis of a large set of HIV-1 sequences revealed that all three regulatory features of the 59ss region are highly conserved in different group M viruses, which supports an important function during virus replication. Modulation of the splicing frequency reduces HIV-1 replication and, as previously discussed (Mueller et al, 2015), the HIV-1 splicing process is an interesting target for antiviral therapy. …”

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confidence: 99%

“…DNA affect splicing efficiency when tested in the context of a destabilized SD hairpin structure (Mueller et al, 2015). However, two putative SRSF2 sites [SRSF2 (1) and SRSF2 (2) ] are present upstream of the 59ss.…”

mentioning

confidence: 99%

“…The 59ss region can fold a stemloop structure, the splice donor (SD) hairpin, that partially occludes the U1 annealing site and reduces splicing frequency (Abbink & Berkhout, 2008;Mueller et al, 2014). Splicing is also influenced by binding of several SR proteins to splice regulatory elements (SREs) in the SD region (Asang et al, 2012;Mueller et al, 2015). The SD hairpin structure may also influence binding of these SR proteins.…”

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confidence: 99%

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Human immunodeficiency virus type 1 splicing at the major splice donor site is controlled by highly conserved RNA sequence and structural elements

Müeller

Klaver

Berkhout

et al. 2015

Journal of General Virology

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Human immunodeficiency virus type 1 (HIV-1) splicing has to be strictly controlled to ensure the balanced production of the unspliced and all differently spliced viral RNAs. Splicing at the major 59 splice site (59ss) that is used for the synthesis of all spliced RNAs is modulated by the local RNA structure and binding of regulatory SR proteins. Here, we demonstrate that the suboptimal sequence complementarity between this 59ss and U1 small nuclear RNA (snRNA) also contributes to prevent excessive splicing. Analysis of a large set of HIV-1 sequences revealed that all three regulatory features of the 59ss region (RNA structure, SR protein binding and sequence complementarity with U1 snRNA) are highly conserved amongst virus isolates, which supports their importance. Combined mutations that destabilize the local RNA structure, remove binding sites for inhibitory SR proteins and optimize the U1 snRNA complementarity resulted in almost complete splicing and accordingly reduced virus replication. Human immunodeficiency virus type 1 (HIV-1) produces a single primary RNA transcript. The full-length transcript functions as RNA genome that is packaged into virions and as mRNA for translation of the Gag and Pol proteins. HIV-1 RNA contains several splice donor (SD) [59 splice site (59ss)] and splice acceptor [39 splice site (39ss)] sites. Differential usage of these sites results in the production of a variety of spliced RNAs that encode the other viral proteins. Splicing has to be strictly regulated for the balanced production of all viral RNAs and proteins.Formation of the spliceosome complex is initiated by binding of U1 small nuclear ribonucleoprotein (snRNP) to the 59ss, which is mediated by the complementarity between the 11 single-stranded nucleotides at the 59 end of the U1 small nuclear RNA (snRNA) component and the 59ss sequence (Freund et al., 2003). The major 59ss located in the 59 untranslated leader of HIV-1 transcripts is used for the production of all spliced mRNAs. As both spliced and unspliced transcripts are required for a productive replication cycle, splicing at this 59ss has to be controlled to prevent complete processing. Previous studies indicated that both RNA structure and sequence elements are involved in this control. The 59ss region can fold a stemloop structure, the splice donor (SD) hairpin, that partially occludes the U1 annealing site and reduces splicing frequency (Abbink & Berkhout, 2008;Mueller et al., 2014). Splicing is also influenced by binding of several SR proteins to splice regulatory elements (SREs) in the SD region (Asang et al., 2012;Mueller et al., 2015). The SD hairpin structure may also influence binding of these SR proteins. In addition, the extent of sequence complementarity between the 59ss and U1 may influence snRNP binding and splicing efficiency.

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HIV-1 splicing is controlled by local RNA structure and binding of splicing regulatory proteins at the major 5′ splice site

Cited by 18 publications

References 59 publications

Characterizing HIV-1 Splicing by Using Next-Generation Sequencing

Characterizing HIV-1 Splicing by Using Next-Generation Sequencing

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Human immunodeficiency virus type 1 splicing at the major splice donor site is controlled by highly conserved RNA sequence and structural elements

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